Project Summary Multicopper oxidases (MCOs) are a large and diverse family of enzymes which couple the one-electron oxidation of various substrates with the four-electron reduction of dioxygen to water. They are important in biological processes and are increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. Human serum ceruloplasmin (Cp) is the most complicated MCO with one trinuclear copper (TNC) center and three type 1 copper (T1Cu) sites. One of the main reasons for the interest in Cp is the mutational disease aceruloplasminemia, which reveals a role for Cp as an essential ferroxidase critical for iron homeostasis. A fundamental understanding of the catalytic cycle of the MCOs has been developed based on the study of Rhus vernicifera laccase (RvL). However, the mechanism of Cp catalysis is still unclear. Preliminary studies reveal that an intermediate of Cp catalysis is different from that of RvL, and the functions of the two additional T1Cu sites of Cp are not well defined. Hence, this proposal focuses on these two important issues regarding the Cp mechanism: 1) The nature of the unique intermediates involved in Cp catalysis; 2) The roles of the two additional T1Cu sites. All the intermediates or mutants involved will be analyzed by a wide range of spectroscopic methods correlated with DFT calculations to define the corresponding electronic and geometric structures of the intermediates and elucidate reaction coordinates. The correlation of Cp intermediates to the well-defined intermediates present in other MCOs will be determined. These mechanistic studies of the Cp will advance the fundamental understanding of the structure-activity relationship of human Cp and provide valuable insight into the pathogenesis of metal-mediated diseases and mechanisms for metal neurotoxicity.